For many years researchers have attempted to perfect a cell culture medium which enabled animal cells to proliferate in vitro. Two general types of cell culture medium have been developed: culture medium supplemented with pituitary extract or serum (Serum-Supplemented Medium: SSM), and chemically defined media without serum (Chemically Defined Medium: CDM). With respect to SSM, the addition of serum to the culture medium introduces a very large number of undefined components. Among these are regulatory compounds, that is to say compounds which regulate cell metabolism. Compounds such as these complicate the study of the regulatory mechanisms which control the proliferation, differentiation and longevity of animal cells. Moreover, in the case of industrial use of established animal cell lines, the presence of serum or of other unknown or poorly defined components creates difficulty in purifying the one or more biological products produced by the cells. In addition, because many biological products cannot be purified, the non-defined composition of SSM leads to prohibitions against human utilization of these biological products by the international and national health authorities (FDA, Ministries of Health, and the like).
One type of SSM which has been used extensively in research is "high" calcium SSM. "High" calcium medium is defined as medium having greater than 0.1 mM calcium. Several researchers have demonstrated the importance of calcium in culture medium. Long term primary cultures of epidermal cells have been difficult to establish and maintain in high calcium medium. "Low" calcium medium is defined as medium having less than 0.1 mM calcium. It has been reported that in "low" calcium medium, although keratinocytes will proliferate, they neither stratify, make desmosomes, nor produce keratins characteristics of their terminal differentiation (1). However, increasing the concentration of calcium in these media induces failure to proliferate, terminal differentiation and sloughing of all normal epidermal cells (2, 3, 4). In the past, a selection with "high" calcium medium has provided an excellent vehicle for assaying for carcinogen or virally altered epidermal cells, as well as the factors involved in the induction of terminal differentiation (2, 5, 6, 7, 8). Nevertheless, "high" calcium SSM is inappropriate for any studies dependent upon concomitant proliferation and differentiation such as, comparing the proliferation potential of normal untreated epidermal subpopulations, or for biological, toxicological, and metabolic studies which may be influenced by undefined components in the medium.
To overcome the various drawbacks presented by cell lines established in "high" calcium SSM, various authors have replaced serum with hormones, electrolytes, and growth factors, such as, insulin, calcium and epidermal growth factor, to create "high" calcium CDM. However, none of these media have been as successful as the "high" calcium SSM in establishing and cultivating primary and secondary cell cultures. The present inventors previously developed a "high" calcium CDM which was used to establish and cultivate epidermal cells from adult mice. This formulation, however, was found to be inferior to a companion "high" calcium SSM with regard to culture longevity (1). Accordingly, a new "high" calcium CDM is needed which overcomes the problems associated with "high" calcium SSM and prior "high" calcium CDM.
A new "high" calcium CDM which is superior or equal to "high" calcium SSM and prior "high" calcium CDM in establishing and cultivating animal cell offers many advantages over prior animal cell culture medium. Among these are: 1) the long term growth and proliferation of freshly isolated animal cells; 2) ease of purifying biological products from the medium for therapeutic applications; and 3) the ability to conduct metabolic, toxicological, and carcinogenesis studies without interference by undefined factors.
The present inventors have developed a chemically defined, "high" calcium animal cell culture medium. The inventive medium now enables researchers to investigate the influence of biological and xenobistic agents on the outgrowth and subsequent longterm maintenance of normal animal cells obtained from explants or primary cultures of dispersed cells. The defined composition of the present inventive medium and its ability to support the growth and differentiation of animal cells in vitro should be useful for a number of studies related to carcinogenesis, toxicology, cell metabolism and pharmacology, for example in a murine model system, that have not previously been possible.